Functional domains of the LIM homeodomain protein Xlim-1 involved in negative regulation, transactivation, and axis formation in Xenopus embryos

Xenopus LIM homeodomain protein Xlim-1 is specifically expressed hi the Spe mann organizer region and assumed to play a role in the establishment of th e body axis as a transcriptional activator. To further elucidate the mechan ism underlying the regulation of its transcriptional activity, we focused o n the region C-terminal to the homeodomain of Xlim-1 (CT239-403) and divide d it into five regions, CCR1-5 (C-terminal conserved regions), based on sim ilarity between Xlim-1 and its paralog, Xlim-5. The role of Xlim-1 CT239-40 3 in the Spemann organizer was analyzed by assaying the axis-forming abilit y of a series of CCR-mutated constructs in Xenopus embryos. We show that hi gh doses of Xlim-1 constructs deleted of CCR1 or CCR2 initiate secondary ax is formation in the absence of its coactivator Ldb1 (LIM-domain-binding pro tein 1), suggesting that CCR1 and CCR2 are involved in negative regulation of Xlim-1. In contrast, while Xlim-l is capable of initiating secondary axi s formation at low doses in the presence of Ldb1, deletion of CCR2 (aa 275- 295) or substitution of five conserved tyrosines in CCR2 with alanines (CCR 2-5YA) abolished the activity. In addition, UAS-GAL4 one-hybrid reporter as says in Xenopus showed that CCR2, but not CCR2-5YA, with its nanking region s (aa 261-315) functions as a transactivation domain when fused to the GAL4 DNA-binding domain. Finally, we show that none of the known transcriptiona l coactivators tested (CBP, SRC-I, and TIF2) interacts with the Xlim-l tran sactivation domain (aa 261-315) Thus, Xlim-l not only contains a unique tyr osine-rich activation domain but also contains a negative regulatory domain in CT239-403, suggesting a complex regulatory mechanism underlying the tra nscriptional activity of Xlim-l in the organizer. (C) 2000 Academic Press.